Telomeres protect human chromosomes from degradation, fusion, and other enzymatic attack. During the replicative cycle telomeres shorten and therefore erode as a function of cell division. This mechanism limits the number of times cells can divide, representing a powerful tumor-suppressive pathway. Cancer cells, in order to acquire immortality, have to circumvent replication-associated telomere shortening and do so by activating either one of two possible telomere maintenance mechanisms. Most cancer cells switch on the telomerase pathway, dependent on the upregulation of the catalytic subunit of telomerase, a reverse transcriptase that specifically elongates the TTAGGG repeats at chromosome ends. Consequently, telomerase activity is a hallmark of most cancer cells and telomerase is the subject of intense research and targeting approaches. A subgroup of cancer cells, frequently of mesenchymal origin, maintains their telomeres in the absence of telomerase activity. These cells manage to avoid telomere shortening by activating Alternative Lengthening of Telomeres pathways (ALT). ALT is an outcome of recombination between telomeres, leading to DNA synthesis and consequently to length gains, avoiding critically short telomeres. While it is known that recombination is required for ALT, it is entirely unclear how ALT is activated, how ALT is maintained and why some tumor types prefer to activate ALT instead of telomerase. Furthermore, evidence is emerging that ALT can be activated as a resistance mechanism to telomerase inhibition, pointing out that both telomerase and ALT have to be understood and inhibited, before the targeting of telomere length mechanisms can become an effective and widely used cancer therapy. Previously no experimental models existed where ALT can be induced in a controlled environment. Progress during the last grant period has provided such models in nematodes as well as in mammalian cells. In the three specific aims of this renewal application, it is proposed to take advantage of these models to further our understanding of ALT activation and regulation. First, the molecular steps of ALT activation in C. elegans will be deciphered and changes in the telomeric complex upon ALT induction will be investigated. Second, the focus will be on RTEL1, a regulator of ALT that emerged from research during the past grant period, as a central factor in ALT maintenance. Third, the hypothesis will be investigated that replication fork stalling leads to a chromatin environment that favors ALT activation. In summary, this proposal is designed to take advantage of models for ALT activation in mammals and nematodes to understand this essential pathway.